Review of Scientific Methods and Findings for: Final Report of The Investigation of Unusual Illnesses Allegedly Produced by Endosulfan

Exposure in Padre Village of Kasargod District (N. Kerala)

Robert P. DeMott1, Thomas D. Gauthier1, Diane J. Mundt2

1 - ENVIRON International Corporation, Tampa, Florida, USA 2 - ENVIRON International Corporation, Boston, Massachusetts, USA

Corresponding Author Robert P. DeMott, Ph.D., Principal Toxicologist ENVIRON International 10150 Highland Manor Dr., Suite 440 Tampa, FL 33610 USA rdemott@environcorp.com

Submitted -- March 27, 2012

Scientific Review NIOH Endosulfan Study

Executive Summary
This report evaluates the scientific methods, conduct, interpretations and conclusion of a report titled Final Report of The Investigation of Unusual Illnesses Allegedly Produced by Endosulfan Exposure in Padre Village of Kasargod District (N. Kerala)conducted by the Indian National Institute of Occupational Health (NIOH). This is a project report of a study regarding environmental and human health conditions in an area near cashew plantations where there has been a history of aerial application of the pesticide endosulfan. The report covers environmental sampling of soil, water, sediment and other materials. It also includes a survey used to obtain information about human health conditions and follow-up testing involving physical examinations and blood sampling for endosulfan and hormonal analyses. The report reaches broad and strong conclusions that human health conditions grouped under three headings (neurobehavioral disorders, male reproductive system abnormalities, and congenital malformations in females) are more prevalent in Padre village (the studypopulation), near the sprayed plantations, than in a reference village approximately 25 km distant where this endosulfan application method was reportedly not used. The report goes so far as to conclude that endosulfan is the most probablecause of the conditions documented in the report. The evaluation of this report covers the study design, the analytical chemistry methods and the relevance of the findings to endosulfan effects. First, a necessary limitation of the study design employed to evaluate human health conditions, termed a survey study, is that it cannot reach conclusions regarding the causes of effects noted. This method is generally accepted as suitable solely for preliminary hypothesis generation. Based solely on the study design selected, ascribing endosulfan as the most probable cause of the conditions recorded is not a scientifically valid conclusion, even; without considering the quality of its conduct and findings. When we do consider the analyses conducted and the interpretations, however, serious limitations and inadequacies emerge. There were serious omissions and errors in the computation and presentation of the analytical results for endosulfan in environmental samples. In fact, there was not even confirmation that the measurements made were actually endosulfan. Lack of confirmation, calibration and quality assurance/quality control information are such that these results would commonly be rejected by environmental regulatory agencies and would not be considered reliable for characterizing human health risks for any type of regulatory or judicial action. The survey method as conducted to obtain information regarding human health conditions does not conform to the generally accepted requirements for epidemiological studies and is insufficient for reaching a conclusion that endosulfan effects are observed in Padre village. Substantial sources of potential bias were not prevented or considered in the analysis, and then were not discussed in the report as possible sources of uncertainty and limitations. The comparisons were frequently simplistic and, in some cases, presented in ways that served to mask or overlook information that would have weakened the conclusion of adverse effects occurring more prevalently in Padre village. The epidemiological results obtained and the

Scientific Review NIOH Endosulfan Study

analyses conducted also to not meet the standards commonly required for studies used to support regulatory or judicial action. The effects reported to be linked to endosulfan are not biologically plausible as toxicological responses to the low, background levels of endosulfan found. The congenital conditions highlighted for male reproductive effects, in fact, are not even found in excess among the boys from Padre village. And, the measurements characterized to reflect neurobehavioral disorders are not generally accepted as direct markers of such effects. The interpretations stretch highly uncertain and highly subjective tests into such serious terms as disorders, abnormalities,and malformations.The results do not support the presence of elevated rates of such conditions in Padre village. The analyses and interpretations do not meet generally accepted scientific standards for establishing chemical exposures as an explanation for purported health effects. Finally, there were significant and serious discrepancies in some cases between the raw data obtained through a Right to Information request, the presentations made in the report, and the subsequent presentations made in a journal article published from the study. Sample results were excluded, transcription errors were made and numbers of subjects were changed in ways that served to make the conclusions of the report and the journal article in particular stronger. In conclusion, the NIOH Report cannot be used to draw a causal connection meeting generally accepted scientific standards between endosulfan exposure and various reported symptoms and outcomes because of the limitations of the design, the uncertainties and inadequacies of the analyses, and the lack of concordance between the reported findings and actual adverse health conditions.

Scientific Review NIOH Endosulfan Study

1 Introduction
This report provides a critical review of the study prepared by the National Institute of Occupational Health (NIOH), Indian Council of Medical Research, titled Final Report of The Investigation of Unusual Illnesses Allegedly Produced by Endosulfan Exposure in Padre Village of Kasargod District (N. Kerala)(hereafter referred to as the NIOH Report ).

1.1

Background

The NIOH Report was prepared in response to several reports in the press of unusual diseases in residents of small villages in the Kasargod district of Northern Kerala. The villages are located below hilltop cashew plantations that have been treated for control of tea mosquitoes by aerially spraying with endosulfan insecticide two to three times a year for over 20 years (NIOH, 2002; Saiyed, 2003). At the request of the Indian Council of Medical Research (ICMR), a three-member team from NIOH visited the area in August 2001 and recommended following up their visit with an epidemiological study to investigate the prevalence of disease in school children from the targeted population and a nearby control population. The field study was conducted from September 24 to October 7, 2001 with the following objectives (NIOH Report page 5): To confirm the reported disease pattern in the exposed populations and evaluate the magnitude of the problem by comparison with control populations through a well designed epidemiological study. To search for etiological factors if the exposed populations show abnormal disease patterns and generate a hypothesis. To confirm the presence of endosulfan residues in environmental and biological samples and estimate their levels. An initial draft (the First Report) was promised by December 2001 and a final version of the report (the NIOH Report) was published in July 2002. The final NIOH Report included additional analyses of drinking water and soil samples collected in June, 2002, after the initial report.

1.2

Information Sources

In completing our review we relied in part on information provided in: The NIOH Report (NIOH, 2002); An initial draft of the NIOH Report (hereafter referred to as the First Report )(NIOH, undated); and A companion paper published in Environmental Health Perspectives (Saiyed et al., 2003) along with comments on the paper (Abraham, 2004; Indulkar, 2004) and the author response (Saiyed, 2004) published in the same journal. s

Scientific Review NIOH Endosulfan Study

In addition, a limited amount of information was provided in response to the request by Mr. B. Mallesham under the RTI Act 2005. The RTI response included 100 pages of material including: 93 pages of Gas Chromatography-Electron Capture Detection (GC-ECD) output corresponding to 2 soil samples analyzed on May 30, 2002 (Soil001.CHI and Soil002.CHI); 2 water samples analyzed on December 13, 2001 (Water002.CHI and Water003.CHI); 1 blank sample analyzed on November 21, 2002 (Blank001.CHI); and 4 standard samples analyzed on October 29, 2001 (VK013.CHI and VK014.CHI), October 31, 2001 (VK017.CHI) and December 6, 2001 (Std001.CHI). 1 laboratory notebook page (unsigned and undated) describing the extraction and cleanup procedure for analysis of endosulfan in soils. 6 partially masked laboratory notebook pages (unsigned and undated) with limited standard and sample information.

Scientific Review NIOH Endosulfan Study

2 Comments on Analytical Data Quality

The NIOH Report presents the results of analytical chemistry testing used to measure endosulfan concentrations from samples of soil, sediment, water and human blood. The report uses these results to make comparisons between the environmental conditions in the Padre village area, hypothesized to be impacted by aerial applications of endosulfan, and a reference area (Meenja Panchayath village) approximately 25 km away where this type of application was reportedly not used. The report uses the results from blood samples to make comparisons between children from the study versus reference areas. These analytical data are, thus, the critical foundation upon which the comparisons attempting to link endosulfan to the observations in Padre village depend. In our review we identified a number of issues in the way the analytical chemistry tests were conducted and interpreted that affected the quality and reliability of the analytical data. These issues were of a nature and extent that preclude the analytical results being considered valid according to the standards required by governmental regulatory agencies. The results are also unreliable to the extent that they do not meet the generally accepted standards for use in guiding scientific interpretations of environmental or public health conditions. These issues include: Selected data have been excluded from the NIOH Report without explanation. Information obtained from NIOH under the Right to Information Act reveals inconsistencies between the raw data compared to the summary results presented in the NIOH Report. The reporting of endosulfan in water and soil at concentrations well below the minimum detection limit of 1 to 3 ppb and the large numbers of peaksreported in the blank and standard reference samples suggests that random peaks due to electronic noise were routinely misinterpreted as endosulfan peaks. Chromatographic peak identification is based solely on retention time no mass spectrometry confirmation was performed on any of the study samples because endosulfan concentrations were too low to confirm by GC/MS. No calibration data are presented and there is no explanation as to how concentrations in the samples were calculated from instrument readings. Results from QA/QC samples and analyses required for the analytical method are not provided, a condition which frequently results in regulatory agencies rejecting and not relying upon analytical data.

2.1

Excluded Sample Results

Table 1 of the NIOH Report (page 14) provides levels of endosulfan in water samples collected in 2001. Data are presented for six samples (3 well, 1 suranga, 1 stream and 1 pond); presumably, though not specified to be from the study area. Concentrations ranged from -endosulfan) and the degradation product (endosulfan sulfate). While Table 1 contains data for six samples,

Scientific Review NIOH Endosulfan Study

Annexure 6 of the NIOH Report (page 82) notes that total of seven samples were collected a from the exposed area (Village Vaninagar Padre) and three samples from the reference area (Miyapadavu, Meenja Gram Panchayat). Thus, it appears that selected data, including apparently all of the results from the reference area, have been excluded from the NIOH Report without explanation. Excluding the results from the reference area precludes any comparison between the reference and study areas and any valid scientific representation whether the conditions differ between the areas. Similarly, Table 1-A of the NIOH Report (page 15) provides summary data for endosulfan residues in soil samples collected in 2001. The table indicates that eight samples were collected from the study area and 2 samples were collected from the reference area. However, Annexure 6 (page 82) states that eight soil samples were collected in polyethylene bags from the exposed area (Village Vaninagar Padre) and three samples from the control area (Miyapadavu, Meenja Gram Panchayat). Once again, selected data are omitted from the NIOH Report without explanation. The fact that only sample results from the reference area were omitted (i.e., the study and reference areas were apparently treated differently) impairs the scientifically interpretations that can be reached. In addition, the description provided in the NIOH Report suggests that the soil samples were likely not collected at locations useful for comparisons between groups of people from the study village compared to the reference area. The soil sampling locations described for the study area were not in the village itself, where the children reside, but from the cashew plantations 750 meters or more upslope from the village (page 12). The specific location relative to the village is not described for the reference area, but there was reportedly no endosulfan spraying in this area anyway. Comparisons between soil results from the plantations, in the one case, versus the village, in the other case, are not reflective of the potential exposure differences for school children.

2.2

Raw Data from RTI Release Not Matching Results Presented in Report

Raw data obtained from NIOH under the Right to Information Act, 2005 indicate that the summary statistics for endosulfan levels in soil (mean and standard deviation) presented in Table 4 on page 18 of the NIOH Report contain numerous calculation errors leading to inaccurate conclusions as itemized below: -endosulfan concentration reported for the study area (0.274 ppb) in Table 4 -endosulfan concentration measured for the reference area. The -endosulfan concentration reported for the study area should be reported as 0.222 according to the raw data. Note that if the values from the raw data were presented in the table, the soil sample results from the study area (0.222 ppb) would be shown to be lower than the results from the reference area (0.274 ppb). Currently, the report refers to this table to substantiate the statement that levels were higher in the study area as compared to reference area (page 16).The raw data contradict this conclusion.

Scientific Review NIOH Endosulfan Study

Accordingly, total endosulfan concentrations in the top layer of soil, which is the layer where the greatest potential exposures would occur, are also reported inaccurately. According to the raw data provided by NIOH, the mean total endosulfan concentration in the upper soil layer in the study area was 0.242 ppb compared to a mean concentration of 0.303 ppb in the reference area. Again, the concentration found in the study area for total endosulfan is lower, not higher than that found in the reference area, similarly contradicting the study conclusion quoted above. For the middle layer of soil, the raw data provided by NIOH show that the mean -endosulfan was 0.104 ppb in the study area compared to 0.184 in the reference area. Contrary to the study conclusion, this difference is not statistically significant (p = 0.15) at the 95% confidence level, and the study area is, again, lower than the reference area. Table 4 has these values switched and incorrectly reports a value of 0.089 ppb instead of 0.104 ppb as derived from the raw data. In total, 16 out of 24 summary statistics reported in Table 4 of the NIOH Report (66% of the values) differed from summary statistics calculated from the raw data sheets obtained from NIOH under the Right to Information Act, 2005 (see Table 1). Additionally, simple editing and oversight review of the report during its production should have revealed the presence of errors and need for careful evaluation. There is no need for access to the raw data to catch that obviously erroneous information is presented in this table. For example, total endosulfan levels are reported to be less than -endosulfan levels in some cases, which is impossible.

Table 1. Differences Between Summary Statistics Reported in Table 4 of NIOH Report and Summary Statistics Calculated from Raw Data
Soil Layer Constituent Top Study -endosulfan -endosulfan Endosulfan sulfate Total endosulfan -endosulfan -endosulfan Endosulfan sulfate Total endosulfan Report Table 4
0.274 0.161 0.0018 0.004 0.025 0.03 0.030 0.18 0.153 0.067 0.002 0.004 0.007 0.012 0.162 0.08

Area

Mean SD Raw Data

0.222 0.133 0.018 0.039 0.002 0.004 0.242 0.161 0.275 0.162 0.002 0.005 0.026 0.039 0.303 0.187

Difference (Yes/No)

Reference

Yes Yes Yes Yes Yes No Yes Yes

Scientific Review NIOH Endosulfan Study

Middle

Study

Reference

Lower

Study

Reference

-endosulfan -endosulfan Endosulfan sulfate Total endosulfan -endosulfan -endosulfan Endosulfan sulfate Total endosulfan -endosulfan -endosulfan Endosulfan sulfate Total endosulfan -endosulfan -endosulfan Endosulfan sulfate Total endosulfan

0.183 0.076 0.005 0.001 0.008 0.018 0.191 0.08 0.089 0.096 ND 0.007 0.012 0.096 0.091 0.128 0.076 ND 0.012 0.028 0.106 0.085 0.0623 0.06 ND 0.0005 0.001 0.062 0.059

0.184 0.077 0.001 0.001 0.008 0.018 0.192 0.089 0.104 0.098 0.001 0.003 0.004 0.007 0.109 0.098 0.128 0.077 ND 0.012 0.029 0.141 0.087 0.086 0.053 ND ND 0.086 0.053

No Yes No No Yes Yes Yes Yes No No No Yes Yes No Yes Yes

2.3

Detection Limits

-endosulfan and endosulfan sulfate are 1, 1, and 3 pg/ml, respectively. A concentration of 1 pg/ml is equivalent to 0.001 ppb (1 part per trillion [ppt]) and 3 pg/ml is equivalent to 0.003 ppb (3 ppt). There is no description as to how these detection limits were determined and the specified levels do not match those stated twice in other locations in the Final and First Report. The levels on page 84 appear to be the erroneous units for several reasons: 1) Elsewhere in Annexure 6 of the Final Report (page 86) the detection limits for analysis of -endosulfan and endosulfan sulfate by GC-ECD are reported as 1, 1 and 3 pg/l, respectively equivalent to 1, 1, and 3 ppb. 2) T -endosulfan and endosulfan sulfate cited in the First Report (page 61) are given as 1, 1, and 3 pg/l, respectively also equivalent to 1, 1, and 3 ppb. 3) Detection limits of 1-3 ppb would be consistent with the method detection limits reported elsewhere for water samples using EPA Method 508. Achieving detection limits in the range of 1-3 ppt (1 pg/ml) with the volumes of material (500 ml for water) used in this study and no chromatographic cleanup step would be extraordinary, and is highly unlikely. 4) Further, the records showing computations of concentrations from serum samples obtained under the RTI request show that a reference standard concentration of 200 ppb was used in these computations. If the GC/ECD method was actually achieving 1-3 ppt

Scientific Review NIOH Endosulfan Study

detection limits, instead of 1-3 ppb, selecting a standard of 200 ppb would have been unusual. There is a clear discrepancy of 1000-fold between the detection limits specified on page 84 compared to those listed on page 86. We have considered the possibility that this discrepancy is just a lack of clarity related to the conversion between the concentration found in the hexane diluent compared to the detection limit for the original sample of serum or water. We have also reviewed information provided in response to the RTI request that suggests the values of 1-3 ppt to be correct due to the mathematical conversion to account for concentration of the extracts from the samples. However, based on the detection limits being twice specified by the authors to be 1-3 ppb, the reasonable expectations of the GC/ECD method, and the chromatograms and information provided in the RTI response, we have concluded that it is more likely that the statement indicating 1-3 ppt on page 84 is in error and that 1-3 ppb (1-3 pg/l) endosulfan in serum or water is more likely the correct detection limit. If sample detection limits of 1-3 ppt were actually achieved with the water samples, this would need to be clearly explained and specified. Throughout the descriptions of the method and detection limits, there is no information at all regarding extraction methods and detection limits for the solid materials sampled (soil, sediment, leaves). Detection limits for these types of materials are frequently less sensitive than those for water samples, however no in-depth comparison can be made and the validity of the soil and sediment results cannot be assessed without a description of the method, amount of material extracted and the detection limits achieved.

2.4

Chromatographic Peak Identification

Annexure 6 to the NIOH Report includes a description of Confirmation Testssing gas chromatography mass spectrometry (GC/MS). Whereas gas chromatography using an electron capture detector (GC-ECD the primary method used to analyze study and reference population samples) relies on retention time for non-specific compound identification, GC/MS provides specific identification of compounds through mass spectral analysis. To attempt to verify the GC-ECD analysis of serum samples from the study population and document that they were reporting results for endosulfan specifically, the authors analyzed standard endosulfan samples and serum samples from an individual poisoned with endosulfan along with serum samples from the study population by GC/MS. However, this approach was flawed because the levels of endosulfan in serum samples from the study population were below the limit of detection for the GC/MS instrument, which was reported to be 100 pg/l or 100 ppb (NIOH Report, page 86). Using this instrument/method, no endosulfan was detectable in the blood samples from the study population via the specific, GC/MS method. Moreover, the chromatography column used in the GC/MS analysis (a 30m x 0.25 mm id DB-5 column) differed from the column used in the GC-ECD analyses (a 60m x 0.25 mm HP5 column) such that endosulfan retention times in the GC/MS analysis (ranging from 28.8 to 32.5 minutes) were much shorter than the endosulfan retention times reported for the longer 60 m

Scientific Review NIOH Endosulfan Study

column used in the GC-ECD analyses (ranging from 38.9 to 67.0 minutes). Because the retention times did not match between the two methods, the GC/MS results could not even serve to substantiate that the peaks measured in the study population samples via GC/ECD were likely to be endosulfan. The confirmation method capable of specifically identifying endosulfan failed to detect this compound in the serum samples from the study population and the endosulfan standard peaks did not match the retention time peaks quantified as endosulfanfrom the GC/ECD analyses of the study population. Note that EPA Method 508 warns in particular about potential interference from phthalate esters presenting a major problem when using an electron capture detector. Phthalates are plasticizers (the serum samples were stored in plastic) and are also extremely common at low, background levels in human blood samples. Because no independent confirmation of peak identification was performed, there is no way to determine if phthalate ester interference was contributing to the GC/ECD signal interpreted to be endosulfan.

2.5

Random Noise Fluctuations

The number of distinct peaksreported in the chromatographic output provided in the RTI response (including chromatographic data for the standard samples) ranged from 309 to 486. Even the blank sample, which is expected to serve as the zerosample for the method, contained integrated areas for 429 peaks Because it is unlikely that the blank contained 429 . contaminating compounds, this situation suggests that the software that serves to identify peaksand measure their area was not calibrated to accurately distinguish real peaks from random electronic noise fluctuation. The frequent reporting of results purported to be endosulfan in water and soil at concentrations well below the minimum detection limit of 1 to 3 ppb (e.g., see data reported in Tables 1, 2, 3 and 4 of the NIOH Report) suggests that random electronic noise may have been routinely misinterpreted as endosulfan peaks. In the absence of independent confirmation of peak identification (performed via either GC/MS or dual column chromatography), there is no way that apparent peaks corresponding to the thousandth of a ppb range reported by the authors can be differentiated as actual responses to a specific chemical versus simply electronic background fluctuation from the detector. For example, endosulfan concentrations measured in water samples collected in 2001 are reported in Table 1 (NIOH Report, page 14). Concentrations ranged from 0.0022 to 0.0667 ppb. These concentrations are well below the reported detection limits of 1 to 3 ppb (currently interpreted to be the correct final sample detection limits see Section 2.3, above) and suggest that the authors may in fact be quantifying random noise fluctuations rather than true endosulfan -endosulfan levels were greater than -endosulfan levels, which -endosulfan is the predominant isomer detected (for example see page 45 of the NIOH Report). This further suggests that the peaks -endosulfan may have been neither and reflect instrument electronic noise. Table 2 of the NIOH Report (page 16) presents levels of endosulfan in drinking water. There are no units presented in the table, however by comparison to other tables and the First Report,

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it appears likely that the Report is listing values in ppb. Some values are listed as low as 0.0005 and 0.0004. Values of 0.0004 and 0.0005 ppb are below the minimum detection limits reported in Annexure 6. Similarly, in Table 4 (page 18), which contains a summary of endosulfan levels in soil samples collected in June 2002, mean endosulfan levels are also reported below the detection limit.

2.6

Calibration Data

No mention is provided in the report regarding the method used to calibrate the GC/ECD results, in other words, the calculations needed to convert retention peak areas to concentrations of endosulfan. Calibration curves are not provided in an Annexure to the report. Accordingly, there is considerable uncertainty as to how the sample concentrations were determined. No calibration curve demonstrating linearity of the detector response is presented. There is no indication as to how many standards were used to construct the calibration curve. The lowest standard on a calibration curve should be approximately ten times the method detection limit (i.e. around 10 ppb for this study); however, no standard in this range was apparently run and the lowest standard used was apparently much higher, i.e., 200 ppb. While a multi-point calibration curve may have been generated at some point, the 6 partially masked laboratory notebook pages suggest that calculations of sample concentrations were actually made using single-point calibration with a 1 ppm standard. To be reliable, among other conditions, single-point calibration should be done using a standard concentration that is within 20% of the expected sample concentration. In this case, the study reported calculated concentrations in the sub-ppb range, thousands to hundreds of thousands of times lower than the single standard (1 ppm) that was used for calculations. Calculations based on this type of extreme extrapolation are highly uncertain and such extrapolation would not be acceptable for typical regulatory agency evaluations of analytical data.

2.7

QA/QC Data

No quality assurance/quality control (QA/QC) data are presented to assess the quality of the analytical data presented in the NIOH Report. Annexure 6 provides descriptions of the methodologies used for analysis of endosulfan residues. The method for endosulfan analysis is said to be based on EPA method Section 5, A, (3), (a) but no actual reference is included and this terminology appears to reflect some type of typographical error since a method number is omitted. The method used is actually similar to EPA Method 508 Determination of Chlorinated Pesticides in Water by Gas Chromatography with an Electron Capture Detector which also

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involves liquid/liquid extraction with methylene chloride and analysis by GC-ECD, as done in this study. 1 Results obtained from QA/QC samples are required to evaluate the accuracy and precision of environmental data collected in a study. Method blanks, field blanks and equipment blanks are used to evaluate issues with contamination. Laboratory duplicates, field duplicates and matrix spike duplicates are used to evaluate the precision of the analyses. Surrogate spikes, matrix spikes and external reference standards are used to establish the accuracy of the results. No such data are presented in the NIOH Report and there are no indications in the report, annexures or lab notes provided that such QA/QC testing was completed. No blank data are presented in the report. There is no indication that surrogate spikes were used in the analysis. No matrix spike/matrix spike duplicate results are presented in the report. There is no indication that any duplicate analyses were performed to assess measurement reproducibility. Without presentation of these QA/QC data, there is no way to affirm the reliability of the data reported in the study. If such QA/QC analyses were not conducted, the study would not meet the generally accepted scientific standards for environmental chemistry analyses. In this case, the results would be rejected and not considered usable for scientific interpretations according to the QA/QC requirements that apply for the U.S. EPA method employed.

1 U.S. EPA. 1995. Method 508 Determination of Chlorinated Pesticides in Water by Gas Chromatography with an Electron Capture Detector Revision 3.1. Edited by J.W. Munch. National Exposure Research Laboratory, Office of Research and Development, Cincinnati, OH.

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3 Survey Design Comments

The NIOH Report specifies that one of its objectives is to evaluate disease patterns through a well designed epidemiological study (page 7). However, the study described in the report is, in fact, a simple survey of children in two populations, one from Padre village, near the plantations sprayed with endosulfan and another from a reference village where such spraying had reportedly not occurred nearby. The study was implemented rapidly, over approximately 2 weeks in 2001, and involved numbers of participants that ultimately proved too small to provide useful numbers of the conditions reported. Under generally accepted methods for the design and conduct of epidemiological studies 2, surveys can only serve as preliminary tools to generate hypotheses and do not serve as a basis for reaching conclusions about the causes of health effects. The study by its own design cannot serve as the basis for conclusion regarding an etiological link (causation) for endosulfan producing the conditions observed. Even for the modest goal of hypothesis generation, the design and conduct of the study presented in the NIOH report was not adequately reliable to produce scientific findings suitable for regulatory or judicial uses or action. Similar to the analytical chemistry results serving as the foundation upon which comparisons of environmental conditions rest, the survey and follow-up testing are the foundation upon which the entire interpretation of potential human health effects relies. And, similarly, this foundation is inadequate for the uses to which it is stretched. The validity and strength of epidemiological study results depend on the study design, data quality and completeness. Factors determining the quality and usefulness of epidemiological studies include the ability to avoid bias, control for potential confounding variables, and including sufficient numbers of exposed and non-exposed cases to limit imprecision due to small numbers. Issues relating to these factors in the NIOH study include: The study design is a survey, which cannot be used to determine causation, and the strength of the statistical analyses presented cannot be validated because details on the population base and how the participants reflect their respective communities are lacking. The study fails to account for or discuss the numerous sources of potential bias. The study fails to address, account for or discuss known and suspected causes of the numerous outcomes evaluated, known as confounding variables. Small percentages of surveyed populations participated in some tests conducted for the study.
2

Kleinbaum, Kupper and Morgenstern, 1982; Epidemiological Research: Principals and Quantitative Methods. Van Nostrand Reinhold; Rothman, Greenland and Lash, 2008; Modern Epidemiology, Lippincott Williams & Wilkins

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3.1

Study Design Considerations

The NIOH Report fails to describe how the actual participants were selected, or to provide the total number of subjects in each village who would be eligible for inclusion. Without this information, it is not possible to consider whether those included are representative of children in the corresponding villages. Thus, it is not possible to ascertain the extent to which selection forces were operating, resulting in the 619 and 416 in the study and referent groups, respectively (page 8). Limited information on the methods of recruitment is provided, and whether these efforts were comparable and similarly extensive in each location is not discussed. The introduction of the report describes anecdotal reporting of cases of illness in this area, which would be expected to sensitize the population in the studyvillage as compared to the referencevillage. This situation can lead to differential reporting and participation in a study, thus biasing findings due to over-reporting in the studyvillage. If different methods were used to recruit participants between the communities and if the studyvillage was aware of what was under investigation, any analyses would be subject to potential bias. Methods to survey/interview participants are described to include staff training (page 9). However, the staff was apparently not blindedto the exposure status of the two groups, and since the assessments included subjective characterizations, the extent to which observer bias has been introduced is not known. Given that abnormalitieswere subjectively identified by the staff, and no criteria are provided that define major abnormalities,the potential for intensive scrutiny or differential inclusion of children in the studygroup is high. This could lead to the appearance of a greater number of abnormalities in the study group. This factor is particularly relevant with regard to the Sexual Maturity Rating (SMR) scoring for male subjects. While Annexure 3 contains tables presumably used to train and guide project staff on the specific criteria and corresponding scores for girls (page 70-71), no such tables are provided for boys. The lack of specific and consistent grading categories for boys increases the likelihood of differential scoring by different examiners and increases the potential of observer bias. Also, the proforma questionnaire for boys calls for two SMR scores to be recorded, one for pubic hair and one for external genitalia and testes (page 65). No line is provided for a separate discrete score corresponding to stage of penis development. However, in the analyses that were presented in the report, separate SMR scores are presented in the following categories: pubic hair, penis, and testes. Given a direction on the questionnaire form to score external genitalia and testescollectively, there is substantial potential for errors and uncertainty attempting to separate this into separate scores for penis versus testes development later in the analysis phase, particularly if the observers were not provided a table with the scoring criteria. A questionnaire is provided in the NIOH Report; however, the report states (page 9) that the parents were interviewed in one of four local languages. The possible misunderstandings and

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misinterpretations resulting from translation of the health questions into local languages from English may in fact be substantial, especially if there is no comparable term for the symptoms listed on pages 60 and 61. Bias can be introduced by the interviewer attempts to explain or s translate from English, and if these explanations are more detailed or extensive for the study group, a bias toward over-reporting can result. However, the report does not indicate how many participants were issued the questionnaire in each of the languages, nor does the report discuss any of the challenges involved in the translation or understanding of the symptoms in each of the languages.

3.2

Confounding Factors and Alternate Interpretations

A significant confounding variable in this study important to a number of the analyses presented is the inherent age difference between the groups of children from the two villages. The average age of the reference population is about 10.5 years, compared to 12 years in the study population (Table 7). In the pre-pubescent and pre-teen years, there can be significant differences in growth, maturation, development, and learning that are simply a function of how children develop. Lack of discussion of how an age difference would be expected to affect such outcomesis a serious oversight in qualifying the interpretation of the results presented. The report also failed to provide data describing the ethnic differences among the populations studied, which could reflect real differences in socio-economics, nutrition, or genetics that might affect healthmeasures if not considered in the analysis. The limited narrative discussion of this topic does not provide sufficient information to exclude cultural factors as playing a role in the differences subsequently found in the survey. The suggestion that because differences in height and weight were not statistically significant between the groups, the nutritional status is comparable (page 21) is not substantiated, and cannot be assumed to be a non-factor in other outcomes assessed. Also, the lack of significance for the reported differences in height and weight is likely due to the statistical method chosen and failure to stratify the groups by age. This factor is discussed further below (Section 4.3). Other possible confounding variables include differential diets, hereditary (genetic) factors associated with certain ethnicities, quality of teaching between the schools, and family factors that might differentially influence a child performance in school. The lack of acknowledgement s and control for these and other confounding variables is one of the most serious flaws in the study. The presentation of neurobehavioral problemsis similarly lacking a discussion of alternative explanations for the results reported (Table 9). Again, the children in the two study populations are of different ages, and thus, the comparison of learning disabilitiesand class retention have not considered the affect of age, differences in school teaching standards, possible bias on the part of teachers identifying more disabilitiesamong the study village, or family and home factors that may be affecting learning and classroom behavior. The lack of consideration of age and other extenuating factors and possible teacher bias are also ignored in reporting findings in Tables 10 and 11, the latter based on very small numbers

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3.3

Size of Study Groups

Most of the simple tabulations of findings presented in the report show that less than the full number of participants in each group responded or participated. While incomplete participation and failure to complete multiple components of a study occur frequently, this limits the interpretation of what is presented. Also, when findings are only available for small percentages of study participants, the uncertainty and potential bias become much greater. The report does not report or attempt to evaluate the scale of uncertainty that resulted from these factors. Uncertainty analyses are a common component of epidemiological studies and methods to evaluate the statistical limitations resulting from the low response rates for various endpoints were readily available. Examples include: Chromosomal analyses presented for 2% of reference group and 5% of study group. Serum samples obtained from 26% study group and 20% reference group (page 10). Prevalence of seizure disorders presented for 41% reference girls and 42% study group girls (Table 12). There is additional potential for selection bias as the children for whom blood samples were taken (page 10) or sexual maturity examinations done are a small sub-set of each study group (Tables 16 and 18). Any interpretation of comparisons of these small subgroups is not meaningful, and cannot be assumed to represent the whole eligible study population. Another issue creating uncertainty related to the selected sub-groups for certain tests is the difference in participation rates between the study and reference populations. When differing proportions of groups choose to participate, there is the potential that it is occurring because of a desire to self-select, either for or against participation. Large differences in participation rate include: IQ surrogate testing 57% reference group participation compared to 82% participation from the Padre village study population (Table 10). Sexual maturity rating examinations70% participation from boys in the reference village compared to 53% participation from boys in the study group (Table 18). The numbers of children who are compared in the various tables are not sufficient to draw causal conclusions. The analyses are simplistic and do not all represent the full number of participants in each evaluation. For example, Table 14 includes less than half of both the study and referent populations; there is no basis to conclude the majority of girls for whom there is no information are in fact distributed in the same way as what is given. The results that are presented stratified by age include findings in some age groups that are too small to be meaningful. Table 14, for example, presents results for the age distribution of menstruating girls showing higher numbers and proportions of girls menstruating among the older girls from Padre village. However, only eight 15-year old girls were included from the

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reference village, compared with 23 girls of this age from Padre village. Thus, each individual girl from the reference village has a larger influence on the statistics compared to the Padre village group. With this type of sample size difference, the status of just a couple of girls in the reference group has large effect on the apparent difference between the villages. Also, the statistic presented, an odds ratio, is dependent upon the groups being compared not having such confounders present. Since the odds ratio compares girls from the reference village that are younger on average than the Padre village girls, the effect of age can influence comparisons that have been purported to be affected by environmental factors. In another example, results of chromosomal analyses presented in Table 22 include 8 reference and 29 or 21 study participants (for two different measurements). Thus, comparisons cannot be reasonably characterized as being reflective of the two overall populations results are provided from less than 2% of the reference population versus 5% of the study population. With such a small number from the reference population, the lack of chromosomal abnormalities among these few children is not a useful basis for comparison. Also, the report states may It be noted that the chromosomal abnormalities like dicentric chromosome and chromosome exchange were observed in two each of the study subjects. Drawing note to this observation implies such small numbers are significant to interpretations when such minimal observations are not generally accepted as scientifically relevant.

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4 Comments on Endosulfan Exposure and Effects

One of the specified objectives of the NIOH study was to evaluate etiological factors that could explain the health conditions observed in the study population; in other words, to evaluate potential causes. There are specific generally accepted requirements for evaluating potential causation in toxicological or epidemiological studies and these include the requirement that purported effects be biologically plausible. In the context of a survey, this means that observed conditions being evaluated must be consistent with the biological responses that occur from a putative cause, in this case endosulfan. Also, evaluating causation specifically requires consideration of possible alternate explanations for observations. We have evaluated how the conditions and health effects reported in the NIOH Report relate to the toxicological characteristics of endosulfan and whether the findings reported can substantiate an etiological (causal) role for endosulfan. We have identified issues with the relevance of the survey and follow-up testing of a nature and degree that the report does not meet the standards typical for studies establishing causation for use in regulatory or judicial actions. The issues include the following areas: The endosulfan levels measured in blood samples from the study population in Padre village were within the expected range reported in other studies, making this a weak candidate cause for conditions in the village. Age differences between the study group and reference group are an obvious potential cause of differences in hormone levels that was not fully addressed. Observations purportedly associated with neurobehavioral conditions and congenital conditions are insensitive and not toxicologically relevant.

4.1

Endosulfan Levels Consistent With Background

Endosulfan levels in blood samples from the study group are typical of general background levels found in other populations exposed through routine environmental and dietary sources (i.e., no specific source such as aerial spraying). In and of itself, this circumstance makes endosulfan a difficult candidate to establish as the cause of conditions in Padre village, since there is no indication that exposures are substantially different than experienced elsewhere. Additionally, the computation of values from the reference population generated levels that are unusually low in comparison to expected background levels. Together, these circumstances suggest it is more likely there is something out of the ordinary about the computed values from the reference village than the results from Padre village. Comparisons made between these reference group results and the results from Padre village are, thus, weakened by the uncertainty inherent in the former being inconsistent with other scientific studies. Endosulfan testing results were presented for blood serum samples collected from sub-groups of the children studied in each village. These sub-groups were not selected randomly and are stated to be an outcome of the willingness of parents and children to consent to blood sampling. Results are reported for approximately 1 out of 5 children studied in the reference village and 1

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out of 4 children studied in Padre village. The difference in participation rates suggests that parents in Padre village were more likely to consent, potentially because they were more sensitized to concerns regarding endosulfan. This highlights the potential for selection bias affecting the outcome of comparisons based on the serum sample results. The NIOH Report references Table 5 stating that endosulfan residues were found in 85% and 78% of female and male subjects, respectively of study area; whereas they were found in 34% and 29% of female and male subjects in the reference group (page 16). First, there appears to be a typographical error in Table 5, because the percentages stated to be from the study area are actually shown in the table as being from the reference area and vice versa. In this case, based on comparison of the subgroup sizes subsequently reported in the published version of the study (Saiyed et al., 2003), it appears that the error is in the table heading, as opposed to the text explanation. Additionally, this statement fails to make clear that the percentages do not actually apply to the overall subject groups, but only the non-randomly selected subgroups. Thus, endosulfan was not actually detected in 85% of the 619 subjects from Padre village, rather in 85% of the 97 children who provided blood samples. The NIOH Report presents serum levels of -endosulfan, -endosulfan and endosulfan sulfate grouped by gender of the children (Table 6). No units are shown or mentioned in the text, however, by comparison to the published version (Saiyed et al., 2003) it appears that they are likely presented in parts per billion. Combining the males and females, the average result for the group from the reference village is approximately 1 ppb, while the overall average for the study group from Padre village is between 9-10 ppb. Interestingly, it is the results from the reference group that appear unusual compared to other studies. Because of its wide usage in parts of the world, there are routinely found background levels of endosulfan in human blood, even in the absence of specific exposures such as the aerial spraying being investigated around Padre village. Results from a study conducted in four Punjab villages found average levels of -endosulfan and -endosulfan of approximately 5 ppb in human blood samples (Mathur et al., 2005). A study in Spain reported an average total of approximately 9 ppb for -endosulfan, -endosulfan and endosulfan sulfate combined in umbilical cord blood from newborns, and even higher levels of other endosulfan metabolites not accounted for in the NIOH study (Cerrillo et al., 2005). These results suggest that background levels should have been expected to be in the 5-10 ppb range where exposures were general environmental and dietary sources of endosulfan. The results from the Padre village group appear to be within the range of routine background endosulfan levels found in other populations. The reported values for this village do not stand out as being obviously elevated due to exposures from the aerial spraying patterns on the plantations in the area. Conversely, the reported average values of approximately 1ppb in the reference area appear low relative to expected background and suggest the need to obtain the raw data from these measurements to ascertain how non-detect values were handled in computing the averages. The report does not specify whether averages were computed only using detected values. With

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more than 2/3rd non-detected samples from the reference village, the approach used to deal with censored values is important to consider. Further information suggesting the potential impacts of including samples below detection limits in the analyses emerges by referring to the First Report. Table 2 (page 12) in the First Report provides a listing of individual serum sample results for 22 individuals from Padre village. This version of the report specifies that at that point, 170 children from Padre village had been sampled, but no individual nor summary information is presented for the remaining children. For serum samples, the specified detection limit was 3 ppb for endosulfan sulfate. Three children are listed to have values lower than this (1.57, 2.79 and 2,9 ppb) in the table. The quantification of these results is uncertain if they are below the minimum detection limit for the method. In the Final Report, no individual measurements are provided, just summary information such as the mean, which is influenced by the manner in which censored values (below detection limits) are included. The handling of the reported values below detection limits is not specified. They should have been treated as non-detects.

4.2

Samples Excluded from Journal Publication

The NIOH study is dated 24 July 2002. A journal article presenting some of the data and results from the study, particularly focusing on the results for the males, was submitted for publication on 10 February 2003 (Saiyed et al., 2003). With regard to the blood sample results, there are notable differences in the datasets presented in these two documents. While endosulfan blood levels for 97 boys from Padre village are summarized in the NIOH report, the published article includes results from only 70 boys from the study village. The average total endosulfan level in the published version is 7.47 ppb, compared to 8.71 ppb in Table 6 of the NIOH Report. This suggests that the endosulfan results from the 27 boys excluded from the journal publication were actually a bit higher, on average. The omission of results from these boys is curious since it reduces the apparent elevation of endosulfan in boys from Padre village. This suggests that there was some compelling reason that the results from these 27 boys were not considered suitable for publication, opening up the possibility that data quality issues were recognized between the release of the NIOH Report and subsequent journal publication. In comparison, results from 48 boys from the reference village appeared in the NIOH Report and only 3 of these were excluded in the dataset for the publication, which included results from 45 reference village boys. The outcome that the exclusions occurred to such differing degrees between the groups further suggests there was some factor specific to the detected levels reported in the NIOH Report for the study village that was reconsidered. Also, the published article does not disclose that the results presented are a selected subset of samples from the larger NIOH study or explain the basis for excluding samples. Another unusual quantitative outcome is the reporting of the frequency of detecting endosulfan among the tested samples stated in the journal article, endosulfan was detected in serum samples of 78% of the children in the study group and 29% of the children in the control group (page 1961). These are exactly the same values stated in the NIOH Report and presented in Table 5, albeit apparently reversed. It seems extraordinarily unlikely that the percentages of samples with detected levels of endosulfan could have been identical once 27 study group and

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3 control group boys were dropped. This raises a question as to whether the published statement regarding the frequency of detection is accurate based on the smaller dataset or whether it might have referred to the larger dataset from the NIOH study, which was not disclosed in the article. Presenting summary statistics, such as the frequency of detection, using a different number and group of samples than those described in the materials and methods section of a submitted journal article would be inconsistent with expected transparency in scientific publishing.

4.3

Obvious Alternate Explanations Not Discussed

The data presented in Table 7 show that the girls from Padre Village were older, taller and heavier than the corresponding groups from the reference village. The average age of the study group (12.0 years) is a full year-and-one-half older than the average age of the reference village girls (10.5 years). Particularly at these peri-pubescent ages, such a difference is an obvious alternative factor in hormone levels. Additionally, the girls from Padre village were 19% heavier on average than the girls from the reference village (30.8 kg vs. 25.9 kg). This difference also suggests the obvious possibility that the overall group of girls from Padre village was at a more advanced stage of puberty. The NIOH report does acknowledge the age difference stating, mean age of the study the group is higher as compared to reference population (page 19). However, the report also goes on to conclude that sex-wise distribution is comparable in study and reference groups the (page 19). Such a conclusion in light of a 1.5-year age difference and 5 kg weight difference does not appear adequately supported. The NIOH study reports that levels of Luteinizing Hormone (LH), progesterone and estradiol were higher in the female study group from Padre village compared to the reference village (Tables 26, 29 and 30). This is the outcome that you would expect with such hormones from an older group of peri-pubescent girls and it substantiates that the position stated in the report that the groups from the different villages can be considered comparable is subject to challenge. Both the NIOH Report and the subsequent journal publication (Saiyed et al., 2003) focus extensively on results relating to male reproductive development, particularly testosterone levels and SMR scoring for the boys. However, results relating to both of these parameters are also not adequately considered with regard to alternate explanations. For the SMR scoring, the failure to provide scoring criteria, failure to blindthe observers, and small differences in a subjective score indicate the obvious potential for differences to be explained by observer bias. For the testosterone results, the report and publication acknowledge that age is a controlling factor, but suggest that residence in Padre village is also a statistically relevant factor. However, the statistical testing and information that would allow a reviewer to evaluate the relative importance of these two different causes is not presented. SMR scoring involves assigning a categorical value of 1 to 5 based on certain observable characteristics related to sexual maturity. In the report, the differences reported fall predominantly between scores of 2 and 3 and separate scores for pubic hair, penis and testes

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are presented in tables and figures. The values for penis development cannot be considered reliable because the questionnaire provided to examiners did not even call for a separate score for penis development. It is not clear how the analysts obtained such values. The scores and differences between the study and reference groups for testes development illustrate the subjective nature and high uncertainty of using this parameter. While the criteria employed by project staff for boys were not disclosed in the report or its annexures, a common version of the Tanner SMR criteria relating to development of the testes and scrotum is: SMR Score 2 Enlargement of scrotum and testis, reddening and change of texture of scrotum SMR Score 3 Growth of testes and scrotum Particularly for examiners seeing a patient for the first time, differentiating between whether the boy qualifies for a score of 3 versus 2 is obviously subjective and uncertain. The differences tabulated (Table 18) and plotted (Figure 3) between the study and reference groups show that the Padre village boys aged 12, 13, 14, and 15 scored approximately 0.5 SMR point lower than the corresponding aged boys from the reference village. In other words, the difference reported to be significant corresponds to something about halfway between enlargementversus growthof the testes and scrotum. Also, there are small numbers of boys of each age, ranging from only 5 to 14 with SMR scores of 2 or higher. In the context of these minor differences, unintended observer bias is an obvious possibility that should have been addressed. Providing consistent scoring charts and using examiners blinded to the village of residence for each boy were critical when interpretations depended on such fine distinctions. The NIOH Report states levels of testosterone were lower in the study group as compared to reference population in the same age group (page 37). This statement is not a precise representation of the results in Table 28 because for boys of some ages, the levels were actually lower in the reference group. Looking at boys older than 10, the table shows that the 11 year olds were essentially equivalent between the villages, the reference village boys had a clearly lower average testosterone level for ages 12 and 15 and the Padre village boys had a clearly lower average for ages 13, 14 and 16. And, the 16-year old group from Padre village included only two boys. This type of inconsistent pattern based on small numbers is not a clear indication of effects in Padre village. Statistical analyses intended to help differentiate between the obvious effect of age on testosterone production and a possible effect of location (i.e., village of residence) were apparently conducted. However, the summary information presented is not sufficient to determine the relative contribution of these two factors or to reanalyze the data to confirm the report findings.

4.4

Recorded Observations Not Biologically Relevant

The conclusions presented in the NIOH report specify that the study identified a higher prevalence of neurobehavioral disorders, male reproductive system abnormalities, and congenital malformations in females in the study group from Padre village. Interpreting the

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findings of the study in these terms is inconsistent with both the results obtained and the generally accepted meaning of these terms. The results apparently categorized to indicate neurobehavioral disorders were 1) differential scholastic performance, 2) differential performance in a test to draw a man, and 3) abnormal behavior reported by teachers. None of these parameters are directly or uniquely indicative of a biologically based neurobehavioral disorder. The report states prevalence of arrogant and the aggressive behavior and restlessness were higher in the study group as compared to the reference population (page 22). According to reporting by teachers for the 619 Padre village children, 1.8% of them were arrogant, 1.3% were aggressive and 0.3% were restless. The comparison made is to reporting from the teachers of the 416 reference village children among whom there was not a single restless or aggressive child noted and only one child reported to be arrogant. Such a finding is clearly a difference without meaning in the context of declaring children to have neurobehavioral disorders.Given the awareness of the Padre village teachers of the concerns regarding endosulfan, reporting bias is an obvious consideration, along with some type of motivation to underreport that may be affecting the teachers of the apparently 415 near perfect children from the reference village. These endpoints are not appropriately sensitive to identify actual clinically relevant disorders and are not indicators of endosulfan toxicology. In addition to the reported differences in testosterone levels and SMR scoring discussed above, the other observations apparently interpreted as male reproductive system abnormalities in the report include two conditions reported in Table 12 undescended testes and congenital hydrocele. Two cases of undescended testes were reported among 361 boys from Padre village, amounting to 0.55% of the boys. This is a relatively common condition with a prevalence of around 1% in boys over age 2. Higher rates are seen among newborns, but they commonly resolve prior to age 2. The number of cases seen in Padre village is, thus, not higher than would be expected. Curiously, Table 12 shows the fraction 2/361 and parenthetically 1.55%. This incorrect calculation overstates the percentage by almost threefold and makes the table entry appear higher than the general population rate of around 1%. Congenital hydrocele is another common condition at birth, occurring in approximately 1-2% of boys. Hydrocele refers to fluid accumulation around the testes and congential cases also frequently resolve as the testes complete their descent and the scrotum becomes isolated from the abdominal cavity, allowing the fluid to resorb. The report lists four cases of congenital hydrocele, meaning the condition was present at birth, among 361 Padre village boys. This amounts to 1.1% of the group, again not higher than expected. In the published journal article version of the study (Saiyed et al., 2003), cases of a third condition, congenital inguinal hernia, are included along with undescended testes and congenital hydrocele, even though there is one case in each of the study and reference populations. The article reports a total of 6 cases among these three conditions (omitting one of the hydrocele cases for some reason) and, then computes a prevalence of 5.1% in Padre village (abstract, page 1958) by revising the group size to report that they were observed among 117 subjects (6/117). Changing the denominator in this manner between the report version and

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published version, and adding an extra case to the numerator from another condition for which there is clearly no difference among the study and reference populations, thus computing a higher prevalence rate from the same underlying study, is inconsistent with expectations of scientific publishing. While the conclusions of the report specify differences in female congenital conditions collectively to be important, review of Table 12 shows that the only significant difference between the two villages was noted for the non-specific grouping congenital heart disease. Nine cases were noted among 258 girls from Padre village. Only one case was noted among 183 girls from the reference village. By segregating the girls in this manner the report shows an apparent difference. However, congenital heart diseases in general are not particularly linked to gender. And, among the boys, the prevalence of congenital heart disease is 3 times higher in the reference village than in Padre village according to Table 12. The observations among the boys are, thus, contradictory to the pattern observed with the girls. Since there is no biologically reasonable basis or research suggesting that endosulfan affects cardiac development in opposing manners between males and females, the observations collectively grouped as female congenital conditions are not, in fact, relevant to establishing endosulfan as the cause. This is a case where the difference is more likely a matter of chance related to the small number of reported cases.

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5 Conclusions
The NIOH report draws a series of conclusions including that there is a significantly higher prevalence of neurobehavioral disorders, congenital malformations in females, and male reproductive abnormalities in the Padre village area. The report also concludes that sufficient information has been considered such that endosulfan exposure from aerial spraying in the cashew plantations upslope from the village is the most probablecause of the health conditions documented in the report. These conclusions are not supported by the study design, methodologies, or results of the investigation that was completed. The conclusions outreach the stated objectives of the study, which specify that the survey approach was intended to serve to generate a hypothesis regarding potential etiological factors. This objective was theoretically attainable, however the survey method employed cannot, as a matter of generally accepted epidemiological science, serve as the basis for determining causation from a single environmental factor. The design and methods are suited solely for preliminary hypothesis generation. Therefore, the survey could not, in the first place, support the conclusion reached regarding endosulfan as the probable cause. Review of the Final Report, the First Report, the information provided subsequent to an RTI request and available scientific literature lead us to conclude that this investigation is impacted by analytical limitations; computational errors; reporting and documentation errors and omissions; and inadequately substantiated interpretations to such an extent that it is not scientifically reliable. The uncertainties are sufficient that results from this investigation would frequently be rejected by environmental regulatory agencies and excluded from consideration of regulatory or judicial actions. We recognized serious limitations in the analyses for endosulfan conducted on environmental and blood samples. The failures to provide QA/QC documentation or confirm the presence of endosulfan with a chemical-specific method and the implications of the large number of apparent chromatographic peaksin the blank create uncertainties with regard to whether endosulfan was even the compound detected in some samples with low reported concentrations. The presentation of summary data alone, without appendices containing the full set of results, makes it impossible to recreate the computed means and variances and ascertain how non-detected values were included. The survey approach used to document health conditions is subject to substantial sources of potential bias and did not include steps to either eliminate or specifically recognize the impact of biases. Tests for confounding factors were not included and the simple summary statistics compared are not sufficient to determine the potential interactions among various factors affecting health. The conditions were grouped and categorized such that the seriousness and prevalence of conditions was overplayed. Endpoints that are highly subjective, such as arrogance or aggressiveness reported by a teacher, were represented to be neurobehavioral disorders. Quantitative measurements such as hormone levels were compared using summary statistics that did not account for the obvious and expected effect of age in pubescent subjects.

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Congenital conditions reported as male reproductive effects were actually found among the boys from Padre village at rates below the prevalence expected in the general population. The reporting of the investigation also failed to meet standards of transparency and clarity expected in scientific reports. Sample results were excluded and the number of subjects participating was manipulated between different versions of reporting the same study. Information presented in a followup publication from the study was selected in ways that inflated the apparent prevalence of congenital conditions in Padre village. Summary statistics were computed from varying numbers of subjects without explanation and there were serious discrepancies in some cases between the raw data obtained through a Right to Information request and the numbers presented in the report and publication. In conclusion, the NIOH Report was necessarily limited, as any investigation, by the practical issues of timing, participation and study design. In addition, however, the uncertainties of the investigation were not made clear and its lack of suitability, as a brief, preliminary hypothesisgenerating study, for informing regulatory or judicial actions was not acknowledged. In contrast, the conclusions presented, in particular the finding that endosulfan application is the most probable causative factor for conditions reported, amount to stretched and selective interpretations of the results obtained. The study is not a valid means to support such a conclusion. The report is not an appropriate or complete scientific representation of the study conduct and findings. Substantive corrections, expanded disclosures and further analyses are necessary before the report would meet the expected standards for scientific reporting.

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References
Abraham, C.C. 2004. Endosulfan Effects: Omissions and Flawed Data. Environmental s Health Perspectives 112(10): A538. Arrebola, F.J., J.L. Martinez Vidal and A. Fernandez-Gutierrez. 2001. Analysis of endosulfan and its metabolites in human serum using gas chromatography-tandem mass spectrometry. Journal of Chromatographic Science. 39(5): 177-182. Cerrillo, I., Granada, A., Lopez-Espinosa, M-J., Olmos, B., Jimenez, M., Cano, A., Olea, N., and M.F. Olea-Serrano. 2005. Endosulfan and its metabolites in fertile women, placenta, cord blood, and human milk. Environmental Research 98:233-239. Goyal, R.K. and H.G. Koshia. 2011. Report of the Committee to Evaluate the Safety Aspects of Endosulfan. Report submitted to Department of Health & Family Welfare, Government of Gujarat, Gandhinagar, Gujarat, India. March 15. Gupta, P.K. and R.C. Gupta. 1979. Pharmacology, toxicology and degradation of endosulfan. A review. Toxicology 13: 115-130. Indulkar, A.S. 2004. Endosulfan Effects: Inaccurate Data. Environmental Health s Perspectives 112(10): A538-A539. Mathur, H.B., Agarwal, H.C., Johnson, S., and N. Saikia. 2005. Analysis of Pesticide Residues in Blood Samples from Villages of Punjab. Report of the Centre for Science and Environment, Pollution Monitoring Laboratory. New Dehi. March 2005. National Institute of Occupational Health (NIOH). 2002. Final Report of The Investigation of Unusual Illnesses Allegedly Produced by Endosulfan Exposure in Padre Village of Kasargod District (N. Kerala). Report submitted to the Honorable National Human Rights Commission by Indian Council of Medical Research. Ahmedabad-380016. July 22. National Institute of Occupational Health (NIOH). Undated. Report of The Investigation of Unusual Illnesses Allegedly Produced by Endosulfan Exposure in Padre Village of Kasargod District (N. Kerala) (First Report). Report prepared by Indian Council of Medical Research. Ahmedabad-380016. National Institute of Occupational Health (NIOH). 2010. September 24 Letter Report from P.C. Yadav (NIOH) to B. Mallesham re: Information Provided under the RTI Act, 2005. Saiyed, H., A. Dewan, V. Bhatnager, U. Shenoy, R. Shenoy, H. Rajmohan, K. Patel, R. Kashyap, P. Kulkarni, B. Rajan and B. Lakkad. 2003. Effect of endosulfan on male reproductive development. Environmental Health Perspectives 111(16): 1958-1962. Saiyed, H.N. 2004. Endosulfan Effects: Saiyed Response. Environmental Health s s Perspectives 112(10): A539-A541.

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